This proposal aims to develop a novel, highly-specific targeted therapy for treatment of breast cancer. The long-term objective is to convert the proprietary intellectual property, the mouse monoclonal antibody (McAb) JAA-F11, which targets the pancarcinoma Thomsen-Friedenreich antigen (TF-Ag) to a humanized form for use as an adjunct with conventional therapy to improve outcomes in cancer patients by blocking metastasis and through direct cytotoxicity. The hypotheses are: 1) that humanization of JAA-F11 can yield an antibody of the same biologic and chemical specificity as the original mouse JAA-F11 McAb; and 2) that humanized JAA-F11 (hJAA-F11) can be used therapeutically to create a survival advantage for patients with breast cancers, including triple negative breast cancers, through direct killing and by blocking tumor cell spread. Data indicates that this therapy would be useful in 80% of the breast cancer cases. The overall objective of this Phase I application is to advance translational studies with JAA-F11, through selection of a humanized antibody optimized for chemical and biological specificity and reactivity. The specific aims are; Aim 1: Humanize JAA-F1, producing at least 9 candidates. Humanization will be facilitated by use of data previously obtained by x-ray crystallography of JAA-F11 and computational carbohydrate threading which elucidated the amino acids which interact with the carbohydrate antigen (TF-Ag), and those important for the Gal?1-3GalNAc-alpha restriction of the binding site. Humanization will precede using modified CDR grafting onto the human antibody molecule variable sequence found to be most like the JAA-F11 mouse sequence in BLASTp analysis. The suggested 3 heavy and 3 light chain sequences will be synthesized by GeneArt Synthesis and will be cloned into the plasmid vectors for human antibody scaffolds for IgG1 and kappa light chain (provided by Sherie Morrison). To maximize success we will also obtain 3-9 additional candidates of humanized JAA-F11 from a humanization of antibody supplier. Aim 2: Selection of the hJAA-F11candidates with antigenic specificity most similar to JAA-F11 will be performed using enzyme immunoassays (EIAs) and glycan array analyses. Maximum affinity antibody from those with optimal specificity will be selected by surface plasmon resonance analysis. If none of the antibodies show the TF-Ag alpha specificity required, more of the mouse sequence will be added in the production of new hJAA-F11 candidates. Aim 3: Assessment of the biological specificity and reactivity of candidates will be performed by imaging human tumors with radiolabeled hJAA-F11 in mice and by performing ADCC and CDC cytotoxicity assays with human tumor cells. If the hJAA- F11 is not cytolytic, an antibody-cytotoxic agent conjugate will be produced and tested in Phase II. In Phase II the molecule will be prepared for use in clinical trials following pharmacologic and toxicologic testing. Phase III studies will involve clinical testing. Potential partnering or out-licensing points for pharmaceutical company facilitation of commercialization of JAA-F11 can occur after each completed phase of the overall project.